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Multiparameter precursor analysis of T-cell response to antigen
Abstract
Triggering of the T-cell receptor by cognate antigen induces a variety of cellular events leading to cell proliferation and differentiation. While the plasticity and diversity of T-cell responses have been recognized for a long time, few quantitative studies have been conducted to measure what proportion of specific T cells will enter a given differentiation program after antigen stimulation. In the present study, we analyzed human T cells cultured with influenza-peptide-loaded dendritic cells. We compared three individual methods for assaying the frequency of antigen-specific T cells: ELISPOT, tetramer-binding, and proliferation. The three methods yielded similar but not identical results. In order to study these differences at the single cell level, we developed a multiparameter flow cytometric method, which allows simultaneous analysis of antigen-specific tetramer binding, T-cell proliferation, and cytokine production. Based on these data, we used flow precursor frequency analysis to calculate the proportion of eight different precursor subsets in the original, resting population. We conclude that approximately half of the cells that bound specific tetramers actually proliferated and synthesized IFNgamma in response to antigen. In addition, similar numbers of cells that did not bind tetramer proliferated (but did not synthesize IFNgamma). The method allows for an estimate of the precursor frequency of each functional subset within the initial population. It could be applied to additional markers of function and differentiation, combining all parameters into a description of the complex response potential of a T-cell pool.
... Reduction in fluorescence intensity can be quantified by flow cytometry in conjunction with any of several different algorithms to estimate extent of proliferation based on dye dilution. A major advantage of using flow cytometry and cell-tracking dyes to monitor extent of cell division is that the cells can also be stained for expression of other cell surface or intracellular markers to define lineage, functionality, activation state, cytokine expression, etc. (4)(5)(6). ...
... The exact number of generations that can be followed depends upon the initial intensity that can be achieved without compromising cell viability or function, which varies somewhat with the particular dye and instrument being used (34). An important feature of the dye dilution technique is that division-dependent changes in the expression of cell surface markers (e.g., CD25, CD69, and references 14,46,47) intracellular proteins (e.g., cytokines and references 4,9,11), transgene expression (5), antigen binding (4) or other properties of interest can be readily quantified by flow cytometry. Another advantage of proliferation tracking using dye dilution is that at the end of the experiment it is possible to assign a generation number for each cell and assess the relative frequency of cells in each daughter generation. ...
... At the end of the culture period, the cells are stained for surface phenotype, tetramer binding, intracellular cytokines, etc. and then analyzed by flow cytometry. If cells are to be counterstained for cytokine expression, they should be restimulated with antigen for the last 16 h of culture in the presence of Brefeldin A (4,53). Blood used in all studies reported here was collected from consented healthy donors at Roswell Park Cancer Institute, Buffalo, NY, or Dartmouth Hitchcock Medical Center, Lebanon, NH according to the guidelines and recommendations of each respective institution's IRB. ...
Cell-tracking reagents such as the green-fluorescent protein labeling dye CFSE and the red-fluorescent lipophilic membrane dye PKH26 are commonly used to monitor cell proliferation by flow cytometry in heterogeneous cell populations responding to immune stimuli. Both reagents stain cells with a bright homogeneous fluorescence, which is partitioned between daughter cells during each cell division. Because daughter cell fluorescence intensities are approximately halved after each division, the intensity of a cell relative to its intensity at the time of staining provides information about how many divisions it has undergone. Knowing how many rounds of division have occurred and the relative number of cells in each daughter generation, one can back-calculate the number of cells in the original population (i.e., cells present at the time of stimulus) that went on to respond by proliferating. Using this information, the precursor cell frequencies and extent of expansion to a specific antigen or mitogen of interest can be calculated. Concurrently, the phenotype of the cells can be determined, as well as their ability to bind antigen or synthesize cytokines, providing more detailed characterization of all cells responding to the antigen, not just effector cells. In multiparameter flow cytometric experiments to simultaneously analyze antigen-specific tetramer binding, cytokine production and T-cell proliferation, we found that only approximately half of the cells that exhibited specific binding to influenza tetramer also proliferated, as measured by dye dilution, and synthesized IFNgamma in response to antigen. We expect the advent of new cell tracking dyes emitting from the violet to the near infrared combined with the increasing number of lasers and detectors on contemporary flow cytometers to further expand the usefulness of this approach to characterization of complex antigen-driven immunological responses.
... The vital probes PTIR271, PTIR272, PTIR273, and PTIR274 are membrane-intercalating dyes developed at PTI Research, Inc. (Exton, Pa). Like the visible membrane dyes PKH26 and PKH67, the PTIR dyes label cells by partitioning into lipid regions of cell membranes [11] [12]. However, the PTIR dyes are both excited and fluoresce in the far-red to near-infrared region of the spectrum (Figure 1). ...
... Although permanent expression of GFP or the like is more suitable for very-long-term in vivo tracking, availability of these new FR/NIR dyes will be helpful in multicolor studies. The principle of dye dilution might help estimate the extent of cell division , something that cannot be done with GFP alone [11] [12]. Furthermore, the better tolerance of FR/NIR excitation by cells and fluorescent probes due to lower photon energy and decreased phototoxicity should permit long-term time-lapse imaging. ...
... For this purpose, we used CD40-activated donor-derived B cells instead of donor leukocytes from spleen or blood, as stimulator cells, and calculated frequencies of responding recipient T-cells from division patterns measured by flow cytometric analysis of carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye dilution. CFSE-dilution has a sensitivity similar to MHC-tetramer staining[27][28,29,30], and CD40-B cells are a uniform source of professional APC[31,32,33,34]. The second aim was to determine the kinetics of the direct pathway allo-response after liver transplantation by applying this assay. ...
... Because the width of the intensity histogram of T cells in the parent generation spreads slowly during culture, it is difficult to differentiate cells that have divided once from non-divided cells. Supported by other studies[28,37], we considered cells that had undergone at least two divisions as responders in our data analysis. Only ModFitH plots with a reduced chi-square (which is a measurement for the association of the calculated model and the real CFSE-dilution histogram: or in other words, a measurement for the fit of the model into the dilution histogram) smaller than 5 were included in the analysis. ...
Conventional assays for quantification of allo-reactive T-cell precursor frequencies (PF) are relatively insensitive. We present a robust assay for quantification of PF of T-cells with direct donor-specificity, and establish the kinetics of circulating donor-specific T cells after liver transplantation (LTx). B cells from donor splenocytes were differentiated into professional antigen-presenting cells by CD40-engagement (CD40-B cells). CFSE-labelled PBMC from LTx-recipients obtained before and at several time points after LTx, were stimulated with donor-derived or 3rd party CD40-B cells. PF of donor-specific T cells were calculated from CFSE-dilution patterns, and intracellular IFN-γ was determined after re-stimulation with CD40-B cells. Compared to splenocytes, stimulations with CD40-B cells resulted in 3 to 5-fold higher responding T-cell PF. Memory and naïve T-cell subsets responded equally to allogeneic CD40-B cell stimulation. Donor-specific CD4+ and CD8+ T-cell PF ranged from 0.5 to 19% (median: 5.2%). One week after LTx, PF of circulating donor-specific CD4+ and CD8+ T cells increased significantly, while only a minor increase in numbers of T cells reacting to 3rd party allo-antigens was observed. One year after LTx numbers of CD4+ and CD8+ T cells reacting to donor antigens, as well as those reacting to 3rd party allo-antigens, were slightly lower compared to pre-transplant values. Moreover, CD4+ and CD8+ T cells responding to donor-derived, as well as those reacting to 3rd party CD40-B cells, produced less IFN-γ. In conclusion, our alternative approach enables detection of allo-reactive human T cells at high frequencies, and after application we conclude that donor-specific T-cell PF increase immediately after LTx. However, no evidence for a specific loss of circulating T-cells recognizing donor allo-antigens via the direct pathway up to 1 year after LTx was obtained, underscoring the relative insensitiveness of previous assays.
... The results of previous studies have demonstrated that many healthy, unvaccinated adults show clear peripheral blood T cell responsiveness to influenza, presumably as a result of natural infection by the virus [2,3]. Novak et al. [4] have reported that the influenza-driven proliferation of peripheral blood mononuclear cells (PBMCs) is largely attributable to CD3 + CD4 + CD25 + CD8 Ϫ cells, and Bercovici et al. [5] have shown that peripheral blood CD8 + lymphocytes also proliferate when they are exposed to influenza antigens. Thus, natural systemic cell-mediated immunity to influenza is composed of both T helper and cytotoxic T cell responses. ...
... These responses were evident even though none of the individuals had knowingly had an influenza-like illness within 2 years of the start of the study and were similar in magnitude and kinetics to the response exhibited toward TT. Although it is conceivable that some of the responses observed were those of naive T cells, our findings suggest, as has been reported elsewhere [2][3][4][5], that long-term memory to virus can be restimulated in the systemic compartment. Investigations of TMNC responses to influenza antigens in the absence of vaccination revealed that the organized mucosal lymphoid tissues harbored influenza-specific T cells in all of the individuals tested. ...
We sought to determine whether palatine tonsils (PTs) harbor naturally acquired influenza-specific T cell immunity and whether
routine parenteral immunization with influenza vaccine influences mucosal and systemic T cell reactivity. We demonstrate that
tonsillar and peripheral blood mononuclear cells (PBMCs) proliferate strongly to influenza antigens, suggesting that naturally
acquired immunity exists within both the mucosal and systemic compartments. Influenza vaccination induced significantly stronger
T cell responses in both PTs and blood, in addition to increasing titers of antiinfluenza antibodies in serum and saliva.
Morerapid proliferative responses of PTs after vaccination were associated with a shift from a response involving both CD45RA+ and CD45RO+ T cells to an entirely CD45RO+-dependent response. Interestingly, the ratio of interferon-γ to interleukin-5 was dramatically higher in cultures of PT T
cells responding to influenza than in PBMCs. Our data indicate that parenteral influenza vaccination influences both mucosal
and systemic naturally acquired T cell immunity.
... To assess the effect of the therapy on immunologic function, we assessed tumor-specific CD4 and CD8 precursor levels using a dye dilution proliferation assay (46,47). TCR function was assessed via a redirected cytotoxicity assay (53,54). The dye dilution proliferation assay has a sensitivity of 10 À5 and differentiates the renal cell -specific CD4 + and CD8 + T-cell populations. ...
... The lack of a consistent increase in CTL activity, particularly in patients with clinical responses, may reflect the individual heterogeneity in a functional TCR~signaling apparatus in peripheral blood lymphocytes (11,12,21,32). To isolate a more tumor-specific immune response, we used a dye dilution proliferation assay to measure IFN-g -producing CD4 + or CD8 + T cells in response to the culture with tumor lysate -loaded, autologous DCs (53,54). Thus far, four patients who were tested exhibited an increase in at least one time point in antigen-specific CD4 + T-cell precursor frequency in response to treatment. ...
In patients with progressive malignancy, the natural balance between proinflammatory (Yang) and inhibitory (regulatory or Yin) immune pathways is disrupted and favors cancer-specific immune suppression. Therapy with interleukin 2 (IL-2) can mobilize immune effector cells that recognize and destroy cancer. High-dose IL-2 is the only therapy that has consistently induced complete durable remissions in patients with metastatic renal cell carcinoma (RCC) but only in a few of them. The lack of benefit in most metastatic RCC patients is likely due to the ineffective manipulation of other immune circuits critical in regulating tumor cytotoxic pathways. The limited clinical activity of IL-2, RCC vaccines, and other immune therapies to date leads us to postulate that effective clinical treatment strategies will need to simultaneously enhance proinflammatory pathways and disrupt regulatory pathways. We present preliminary studies in RCC patients to highlight the complexity of the regulatory pathways and our approach to shifting the balance of proinflammatory and regulatory immune pathways using dendritic cell-tumor lysate vaccine followed by cytokine therapy.
... After proliferation, CD62L, CCR7, and CD45RA are gradually downregulated. With the continuous change in phenotype, they differentiate into various T cell subpopulations (34,35). Tscm cells are phenotypically similar to Tn cells but also express molecules not expressed by Tn cells, such as CD95 and CD122 (IL-2Rb). ...
Introduction
Adoption of allogeneic T cells directly supplements the number of T cells and rapidly induces T-cell immunity, which has good efficacy for treating some tumors and immunodeficiency diseases. However, poor adoptive T-cell engraftment and graft-versus-host disease (GVHD) limit the application of these methods. Alloreactive T-cell clones were eliminated from the donor T-cell repertoire, and the remaining T-cell clones were prepared as Tscm for T-cell adoptive treatment to reconstruct recipient T-cell immunity without GVHD.
Methods
The subjects in this study included three different strains of mice. Lymphocytes from mice (C57BL/6) were used as the donor T-cell repertoire, from which the Tscm allo-reactive T cell clone was depleted (ATD-Tscm). This was confirmed by showing that the Tscm was not responsive to the alloantigen of the recipient (BALB/c). To prepare ATD-Tscm cells, we used recipient lymphocytes as a simulator, and coculture of mouse and recipient lymphocytes was carried out for 7 days. Sorting of non-proliferative cells ensured that the prepared Tscm cells were nonresponsive. The sorted lymphocytes underwent further expansion by treatment with TWS119 and cytokines for an additional 10 days, after which the number of ATD-Tscm cells increased. The prepared Tscm cells were transferred into recipient mice to observe immune reconstitution and GVHD incidence.
Results
Our protocol began with the use of 1×10⁷ donor lymphocytes and resulted in 1 ×10⁷ ATD-Tscm cells after 17 days of preparation. The prepared ATD-Tscm cells exhibited a nonresponse upon restimulation of the recipient lymphocytes. Importantly, the prepared ATD-Tscm cells were able to bind long and reconstitute other T-cell subsets in vivo, effectively recognizing and answering the “foreign” antigen without causing GVHD after they were transferred into the recipients.
Discussion
Our strategy was succeeded to prepare ATD-Tscm cells from the donor T-cell repertoire. The prepared ATD-Tscm cells were able to reconstitute the immune system and prevent GVHD after transferred to the recipients. This study provides a good reference for generating ATD-Tscm for T-cell adoptive immunotherapy.
... All patients were confirmed to have no detectable residual disease by computed tomography (CT) scan prior to the initial vaccine injection. Development of immune response was defined as observing significantly greater secretion of interferon γ (IFNγ) (22) or induced proliferation of T lymphocytes (23,24) by peripheral blood mononuclear cells 1 week following the third autologous tumor lysatepulsed DC vaccination, compared to unpulsed DC stimulation. ...
Purpose:
We have previously demonstrated that metastatic colorectal cancer patients who exhibit immune responses to a dendritic cell (DC) vaccine have superior recurrence-free survival following surgery, compared to patients in whom responses do not occur. We sought to characterize the patterns of T lymphocyte infiltration and somatic mutations in metastases that are associated with and predictive of response to the DC vaccine.
Experimental design:
Cytotoxic, memory, and regulatory T cells in resected metastases and surrounding normal liver tissue from 22 patients (11 responders and 11 non-responders) were enumerated by immunohistochemistry prior to vaccine administration. In conjunction with tumor sequencing, the Combined Multivariate and Collapsing method was used to identify gene mutations that are associated with vaccine response. We also derived a response prediction score for each patient using his/her tumor genotype data and variant association effect sizes computed from the other 21 patients; greater weighting was placed on gene products with cell membrane-related functions.
Results:
There was no correlation between vaccine response and intra-tumor, peri-tumor, or hepatic densities of T cell subpopulations. Associated genes were found to be enriched in the PI3K/Akt/mTOR signaling axis (P < 0.001). Applying a consistent prediction score cutoff over 22 rounds of leave-one-out cross-validation correctly inferred vaccine response in 21 of 22 patients (95%).
Conclusions:
Adjuvant dendritic cell vaccination has shown promise as a form of immunotherapy for patients with metastatic colorectal cancer. Its efficacy may be influenced by somatic mutations that affect pathways involving PI3K, Akt, and mTOR, as well as tumor surface proteins.
... As a result, each successive generation in a population of proliferating cells is marked by a halving of cellular fluorescent intensity with excitation/ emission maxima of 492/517 nm, that is readily detected by a flow cytometer. CFDA SE produces a more homogeneous cellular labeling and, consequently, much better intergenerational resolution than other cell tracking dyes, such as the membrane marker PKH26 [6]. Using flow cytometric analysis of CFDA SE labeling, 8 to 10 successive generations of lymphocytes can be resolved [7]. ...
Long term monitoring of live cells in vitro or in vivo presents considerable challenges. Fluorescent tracers are used as one of the most common methods. However, fluorescent molecules usually are limited by cytotoxicity and/or lack of persistence within live cells over sufficient time periods. In this study, we evaluated our newly developed cell tracing/tracking reagents, CellTrac Far Red and CellTracker Deep Red, as novel red laser-excitable fluorescent dyes for flow cytometry and live cell imaging applications. These dyes cross the plasma membrane and covalently bind proteins inside cells, where the stable, well-retained fluorescent label offers a consistent, reliable fluorescent signal without affecting morphology or physiology and also exhibiting low cellular toxicity. CellTrace Far Red was demonstrated to be an excellent probe for cellular proliferation studies using flow cytometry, allowing measurement of up to eight generations of proliferating cells. CellTracker Deep Red was shown to be an excellent reagent for the study of cell movement, location and morphology by labeling and imaging live-cells with fluorescence microscopy. In addition, CellTracker Deep Red has excellent cell retention characteristics with bright fluorescence intensity even after 72 hours of cell staining, with signal retained after fixation, and can be multiplexed with other fluorescent dyes.
... The correlations between numbers of circulating CASTs and functional anti-CMV immunity have been found to be imperfect. The disconnect between enumeration of antigen-specific cells and functional response has previously been shown in flow cytometry studies of an influenza model where it was found that only half of the antigen-specific cells detected by multimers were capable of proliferation or cytokine production (Bercovici et al., 2003). To investigate whether it is not enumeration, but function of the CAST that is essential, intracellular cytokine production assays specifically in CASTs indicate that not all CASTs are functional. ...
In the field of transplantation, flow cytometry serves a well-established role in pre-transplant crossmatching and monitoring immune reconstitution following hematopoietic stem cell transplantation. The capabilities of flow cytometers have continuously expanded and this combined with more detailed knowledge of the constituents of the immune system, their function and interaction and newly developed reagents to study these parameters have led to additional utility of flow cytometry-based analyses, particularly in the post-transplant setting. This review discusses the impact of flow cytometry on managing alloantigen reactions, monitoring opportunistic infections and graft rejection and gauging immunosuppression in the context of solid organ transplantation.
... The DDPA assay has been described and validated in comparison with tetramer and ELISPOT assays. 23,25,26 The intra-assay coefficient of variation for tumor-specific PF values was 10%, and both time points from any one patient were tested in the same assay to eliminate interassay variation from the assessment of treatment-related changes. ...
Patients with glioblastoma multiforme (GBM) are profoundly immunosuppressed and may benefit from restoration of an antitumor immune response in combination with conventional radiation therapy and temozolomide (TMZ). The optimal strategies to evaluate clinically relevant immune responses to treatment have yet to be determined. The primary objective of our study was to determine immunologic response to cervical intranodal vaccination with autologous tumor lysate-loaded dendritic cells (DCs) in patients with GBM after radiation therapy and TMZ. We used a novel hierarchical clustering analysis of immune parameters measured before and after vaccination. Secondary objectives were to assess treatment feasibility and to correlate immune response with progression-free survival (PFS) and overall survival. Ten eligible patients received vaccination. Tumor-specific cytotoxic T-cell response measured after vaccination was enhanced for the precursor frequency of CD4+ T and CD4+ interferon γ-producing cells. Hierarchical clustering analysis of multiple functional outcomes discerned 2 groups of patients according to their immune response, and additionally showed that patients in the top quintile for at least one immune function parameter had improved survival. There were no serious adverse events related to DC vaccination. All patients were alive at 6 months after diagnosis and the 6-month PFS was 90%. The median PFS was 9.5 months and overall survival was 28 months. In patients with GBM, immune therapy with DC vaccination after radiation and TMZ resulted in tumor-specific immune responses that were associated with prolonged survival. Our data suggest that DC vaccination in combination with radiation and chemotherapy in patients with GBM is feasible, safe, and may induce tumor-specific immune responses.
... Typical composition is 71.8 ± 7.1% T cells, 3.3 ± 3.6% monocytes, and 24.7 ± 6.4% B/NK cells as determined by CD45 FITC/CD14 PE/CD3 PerCP staining and flow cytometric analysis (n = 39). 49. Though not further used in the studies described here, the cell pellet obtained after cold aggregation and 1 × g fractionation is an excellent source of relatively pure monocytes. ...
In the second edition of this series, we described the use of cell tracking dyes in combination with tetramer reagents and traditional phenotyping protocols to monitor levels of proliferation and cytokine production in antigen-specific CD8+ T cells. In particular, we illustrated how tracking dye fluorescence profiles could be used to ascertain the precursor frequencies of different subsets in the T-cell pool that are able to bind tetramer, synthesize cytokines, undergo antigen-driven proliferation, and/or carry out various combinations of these functional responses.
Analysis of antigen-specific proliferative responses represents just one of many functions that can be monitored using cell tracking dyes and flow cytometry. In this third edition, we address issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T-cell functions. We summarize key characteristics of and differences between general protein- and membrane-labeling dyes, discuss determination of optimal staining concentrations, and provide detailed labeling protocols for both dye types. Examples of the advantages of two-color cell tracking are provided in the form of protocols for (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay and (b) simultaneous monitoring of proliferative responses in effector and regulatory T cells.
... Doublecolor analysis was performed with CFSE and anti-CD8-PEcy5 using a FACSCalibur (Becton Dickinson). Flow cytometric data files were analyzed with the "Proliferation Wizard" module in ModFit LT Macintosh software [29,30]. ...
The graft-versus-leukemia effect of allogeneic marrow transplantation suggests the dramatic effect of the allogeneic T cell to eradicate malignant disease. Preparation and adoptive transfusion of tumor-specific T cells from HLA-mismatched donors might be expected to circumvent CTL tolerance to the tumor. In this study, a soluble, divalent HLA-A2 molecule was constructed with the Fc part of human IgG1 and was pulsed with a peptide related to melanoma tyrosinase 368-376 [Tyr(368-376) (Tyr)] to form the Tyr/HLA-A2 dimer, which allowed loading onto monocytes via interaction of the Fc and FcR. The HLA-A2-negative (HLA-A2-ve) monocytes loaded with the Tyr/HLA-A2 dimer acted as allo-APC with copies of a single allogeneic epitope. After coculture of the HLA-A2-ve PBLs and autologous monocytes loaded with the dimer, CD8+ cells in the coculture show an obvious proliferation and increased frequency of Tyr/HLA-A2 tetramer-stained cells. The sorted Tyr/HLA-A2 tetramer-positive CD8+ cells display an elevated cytotoxic activity against HLA-A2-positive melanoma cells expressing tyrosinase endogenously (i.e., SK-Mel-5) but little against tyrosinase-negative melanoma cells (i.e., A375). The coculture of PBLs and autologous monocytes loaded with allogeneic peptide/HLA complexes offers a novel approach to expand allo-restricted, peptide-specific CTLs, which might be a potential arsenal for treatment of patients with malignant disease, if the tumor-related epitope were defined.
... Such methods would be useful, for instance in studying the progression of subclinical thyroid dysfunction and the genetic susceptibility to AITD. Multiparameter flow cytometric methods, combining tetramerbinding, cytoplasmic cytokine content and proliferation (Bercovici et al ., 2003) may prove useful in this regard, especially with the current pace of development of major histocompatibility (MHC) class II, as well as class I, tetramers. ...
Recent research in autoimmune thyroid disease (AITD) has largely focused on delineation of the autoantigens and their epitopes, but there is now renewed interest in the immunoregulatory properties of T cells, an understanding of which may explain the emergence of AITD in experimental settings. T cell recognition of autoantigens has shown considerable intra- and interindividual heterogeneity, and a mixed pattern of cytokine production indicates that both the Th1 and Th2 limbs of the helper T cell response are involved in all types of AITD. It is now clear that secretion of chemokines and cytokines within the thyroid accounts for the accumulation and expansion of the intrathyroidal lymphocyte pool, and that the thyroid cells themselves contribute to this secretion. The thyroid cells also produce a number of proinflammatory molecules which will tend to exacerbate the autoimmune process. Thyroid cell destruction in autoimmune hypothyroidism is dependent on T cell-mediated cytotoxicity with the likely additional effect of death receptor-mediated apoptosis.
... We have previously published data that demonstrates the utilitiy and comparability of DDPA to ELISPOT and tetramer methods of determining T cell precursors. DDPA has the additional advantage of providing data on T cell proliferative function [15]. The DDPA is a multi-parameter flow cytometric approach, which uses PKH-26 (Sigma, St. Louis MO), a lipophilic dye stably incorporated into the cell membrane. ...
Granulocyte monocyte-colony stimulating factor (GM-CSF) supports the survival, expansion, and differentiation of lymphoid and myeloid derived dendritic cells (DCs). We hypothesized that systemic therapy with GM-CSF in prostate cancer patients could augment prostate cancer-related immunity and induce clinical response.
Eligible patients were randomly assigned to receive either 125 or 250 microg/m(2) GM-CSF subcutaneously three times a week until clinical progression. Prostate-specific antigen (PSA) T cell precursor frequencies were determined by a flow cytometric method.
We were able to show, for the first time, a statistically significant correlation between pre-treatment PSA level and PSA-specific CD4(+) T cell precursors and a trend between pre-treatment PSA level and PSA-specific CD8(+) T cell precursors (P<0.0001 and P=0.059, respectively).
These results suggest that existent immunity to PSA in prostate cancer patients may be a promising target for future immunotherapeutic approaches to prostate cancer.
... Doublecolor analysis was performed with CFSE and anti-CD8-PEcy5 using a FACSCalibur (Becton Dickinson). Flow cytometric data files were analyzed with the "Proliferation Wizard" module in ModFit LT Macintosh software [29,30]. ...
The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A(426-434)) or a self-peptide (Tyr(369-377)). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A(426-434) pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr(369-377) pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag(77-85)) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%+/-3.72% vs 7.01%+/-0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%+/-0.33% vs 0.05%+/-0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%+/-2.58% vs 6.87%+/-1.01%, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%+/-0.3% vs 0.06%+/-0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.
HLA-A2 is the most common serological HLA type among all ethnic groups. Through advances in DNA typing, more than 800 subtypes of HLA-A2 have been identified, and the existence of heterogeneity of antigen specificity among the HLA-A2 subtypes has been suggested by retrospective analyses of allogeneic transplantation patients and by studies of antigen amino acid structure. However, prior to this study, the antigenicity of a given subtype or the mismatch extent between two given subtypes could not be studied in vitro. Here, we used a modified autologous lymphocyte-monocyte coculture method to reveal heterogeneity of antigen specificity among HLA-A2 subtypes. The coculture was set up with HLA-A2 (non-A*02:01) lymphocytes and monocytes, and the monocytes were coated with an HLA-A*02:01/IgG1-Fc fusion protein (dimer) by high-affinity binding of the IgG1-Fc to FcgRI. Lymphocyte proliferation following coculture indicated that HLA-A*02:01 showed antigenicity against the HLA-A2 (non-A*02:01) subtype. Among the most frequent HLA-A2 subtypes in the Chinese population (HLA-A*02:01, -A*02:03, -A*02:06 and -A*02:07), we identified significant -A*02:01 antigenicity for T cells from -A*02:03 or -A*02:06 but not -A*02:07 individuals. Our findings were consistent with retrospective studies of allograft patients with a limited number of involved subtypes, indicating that this modified coculture method provides a practical and reliable means to study the antigenicity of HLA allele subtypes in vitro.
We report on the development and application of a flow cytometer using a 16-channel avalanche photodiode (APD) linear detector array. The array is configured with a dispersive grating to simultaneously record emission over a broad wavelength range using the 16 APD channels of the linear APD array. The APD detector elements have a peak quantum efficiency of 80% near 900 nm and have at least 40% quantum efficiency over the 400-nm to 1000-nm wavelength range. The extended red sensitivity of the detector array facilitates the use of lower energy excitation sources and near IR emitting dyes which reduces the impact of autofluorescence in signal starved measurements. The wide wavelength sensitivity of the APD array permits the use of multiple excitation sources and many different fluorescent labels to maximize the number of independent parameters in a given experiment. We show the sensitivity and linearity measurements for a single APD detector. Initial results for the flow cytometer with the 16-element APD array and the 16-channel readout ASIC (application specific integrated circuit) are presented.
Routine methods for enumerating antigen-specific T-helper cells may not identify low-frequency phenotypes such as Th2 cells. We compared methods of evaluating such responses to identify tetanus toxoid- (TT) specific Th1, Th2, Th17 and IL10(+) cells. Eight healthy subjects were given a TT booster vaccination. Blood was drawn before, 3, 7, 14, and 28 d after vaccination and peripheral blood mononuclear cells (PBMC) were cultured for 7 d with TT, negative control (diluent), and a positive control (Staphylococcus enterotoxin B [SEB]). Activation markers (CD25 and CD69) were measured after 44h (n=8), cytokines in supernatant after 3 and 7 d, and intracellular cytokine staining (ICS) of proliferated cells (identified by dye dilution) after 7d (n=6). Vaccination increased TT-specific expression of CD25 and CD69 on CD3(+)CD4(+) lymphocytes, and TT-specific proliferation at 7, 14 and 28 d post vaccination. Vaccination induced TT-specific Th1 (IFN-γ, TNF-α, and IL-2) Th2 (IL-13, IL-5, and IL-4), Th17 (IL-17A) and IL-10(+) cells as measured by ICS. TT-specific Th1 cells were the most abundant (12-15% of all TT-specific CD4(+) T-cells) while IL10(+) (1.8%) Th17 (1.1%) and Th2 cells (0.2-0.6%) were less abundant. TT-specific cytokine concentrations in PBMC supernatants followed the same pattern where a TT-specific IL-9 response was also seen. In conclusion, TT booster vaccination induced a broad T-helper cell response. This method of evaluating cytokine phenotypes may be useful in examining the impact of nutrition and environmental conditions on the plasticity of T-helper cell memory responses.
Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.(1-5) Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of: stem and progenitor cell quiescence, proliferation and/or differentiation(6-8) antigen-driven membrane transfer(9) and/or precursor cell proliferation(3,4,10-18) and immune regulatory and effector cell function(1,18-21). Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes(1,2,11). "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins(4,16,18). Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class(2-4,16,18,24). The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are: Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies. Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 10(2) - 10(3) times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used. Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile. Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.
Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834) to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm) was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher (27.8 ± 0.7 μm) than for GFP (20 ± 0.5 μm; p = 0.008; n = 50), corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes (36.4 ± 1 μm from the pleural surface) compared with GFP (30.1 ± 0.5 μm; p = 0.01; n = 50). Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs.
To determine whether an autologous dendritic cell (DC) vaccine could induce antitumor immune responses in patients after resection of colorectal cancer metastases and whether these responses could be enhanced by activating DCs with CD40L.
Twenty-six patients who had undergone resection of colorectal metastases were treated with intranodal injections of an autologous tumor lysate- and control protein [keyhole limpet hemocyanin (KLH)]-pulsed DC vaccine. Patients were randomized to receive DCs that had been either activated or not activated with CD40L. All patients were followed for a minimum of 5.5 years.
Immunization induced an autologous tumor-specific T-cell proliferative or IFNγ enzyme-linked immunospot response in 15 of 24 assessable patients (63%) and a tumor-specific DTH response in 61%. Patients with evidence of a vaccine-induced, tumor-specific T-cell proliferative or IFNγ response 1 week after vaccination had a markedly better recurrence-free survival (RFS) at 5 years (63% versus 18%, P = 0.037) than nonresponders. In contrast, no association was observed between induction of KLH-specific immune responses and RFS. CD40L maturation induced CD86 and CD83 expression on DCs but had no effect on immune responses or RFS.
Adjuvant treatment of patients after resection of colorectal metastases with an autologous tumor lysate-pulsed, DC vaccine-induced, tumor-specific immune responses in a high proportion of patients. There was an association between induction of tumor-specific immune responses and RFS. Activation of this DC vaccine with CD40L did not lead to increased immune responses.
Dendritic cells (DC) are under intense preclinical and early clinical evaluation for the immunotherapy of cancer. However, the optimal culture conditions and route of delivery for DC vaccination have not been established. Here we describe the first human application of DC matured with the bacterial agent OK432 (OK-DC), using a short-term serum-free culture protocol, which generates mature DC from CD14+ precursors after 5 days. These cells were prepared within the framework of a National Blood Service facility, demonstrating that DC represent a product which is potentially deliverable alongside current standardized cell therapies within the UK National Health Service. In vitro analysis confirmed that OK-DC were mature, secreted tumor necrosis factor-alpha, interleukin-6, and interleukin-12, and stimulated both T cell and natural killer cell function. To explore effective delivery of OK-DC to lymph nodes, we performed an initial clinical tracking study of radioactively labeled, unpulsed OK-DC after intralymphatic injection into the dorsum of the foot. We showed that injected DC rapidly localized to ipsilateral pelvic lymph nodes, but did not disseminate to more distant nodes over a 48-hour period. There was no significant toxicity associated with OK-DC delivery. These results show that OK-DC are suitable for clinical use, and that intralymphatic delivery is feasible for localizing cells to sites where optimal priming of innate and adaptive antitumor immunity is likely to occur.
Generation and adoptive transfusion of alloreactive cytotoxic T lymphocytes (CTLs) specific for tumor are expected to circumvent tumor tolerance. Here we describe a novel protocol to raise allo-restricted, peptide-specific CTLs by co-culture of murine splenocytes and autologous macrophage bearing an allogeneic H-2K molecule associated with its restricted peptide (peptide/allo-MHC). The extracellular domains of H-2K(d) were fused with constant domains of murine IgG2a heavy chain to generate a fusion protein (peptide/H-2K(d)/IgG2aFc, the dimer) consisting of divalent TCR-ligands and an IgG Fc receptor type I (FcgammaRI)-reactive moiety. The dimer is able to bind to macrophage (Mvarphi) of H-2K(k) via the interaction of the Fc part with FcgammaRI, and cause the H-2K(k) Mvarphi to be coated with the peptide/H-2K(d) complex. The results show that proliferation of CD8+ cells is enhanced and that the specific-tetramer stained CD8+ cells appear more frequently by co-culture of H-2K(k) splenocytes with the autologous Mvarphi loaded with the dimer. Furthermore, the CD8+ T cells from the co-cultural bulk exhibit an elevated cytotoxicity against a specific target (H-2K(d)-restricted, peptide-specific cytotoxicity), compared with that against the irrelevant targets. This study provides a strategy for preparation of allo-restricted, peptide-specific CTLs, which may add to our arsenal for adoptive immunotherapy to eliminate chronic virally infected or tumor cells.
Antigen-specific T cells may be detected and enumerated by short-term ex vivo antigen-specific stimulation followed by cytokine flow cytometry. Most frequently, intracellular IFN-gamma is used to identify T cells specific for cytomegalovirus (CMV), Epstein-Barr virus or HIV. Some researchers use whole blood, others peripheral blood mononuclear cells (PBMC) in this assay; however, the performance of the two systems has never been directly compared. Blood was drawn from previously characterized healthy CMV-positive donors, and CMV-derived peptides or CMV lysate were used as stimulants. In an initial series of experiments, lithium-heparin was identified as the best coagulant to be used. Dose-response curves were established using concentrations between 0.1 and 40 microg/ml of peptides and between 0.1 and 20 microg/ml of virus lysate, respectively. IFN-gamma-positive T cells were expressed as percent of the reference population, and frequencies measured in whole blood and PBMC were compared. Maximum responses were consistently higher in PBMC than in whole blood and were reached at lower concentrations of stimulant. In several instances, responses identified with PBMC were not at all detected with whole blood. In summary, studies using whole blood in this type of assay are likely to underestimate the frequencies of antigen-specific T cells.
The T-cell receptor provides T cells with specificity for antigens of particular molecular structure (the "epitope"); the T-cell pool in an individual responds to the presence of many different antigenic epitopes, but any particular T cell will respond preferentially to one defined epitope. After stimulation of a T cell by the binding of its receptor to its cognate antigen in the context of a major histocompatibility complex (MHC) molecule on an antigen-presenting cell, the T cell will begin to proliferate and synthesize cytokines. Tetramer binding and the enzyme-linked immunospot (ELISPOT) method have been used to determine what proportion of cells in the T-cell pool can bind to a defined antigenic peptide or will secrete cytokines in response to a particular antigenic stimulation. The method described here uses tracking dyes to determine what proportion of T cells will proliferate in response to stimulation. As a flow cytometric "single-cell" method, it can be combined with tetramer and cytokine staining to determine the precursor frequencies of cells in the T-cell pool able to bind tetramer, to synthesize cytokines, and to proliferate in response to antigen.
CFSE dye dilution analysis and [3H] thymidine incorporation were used side by side to assess proliferative responses of peripheral blood mononuclear cells (PBMCs) after vaccination of renal cell carcinoma patients (n=6) with antigen-loaded dendritic cells. Immune responses against the control antigen keyhole limpet hemocyanin (KLH) were induced in all patients. While [3H] thymidine incorporation revealed a 4 to 977-fold increase in KLH-induced proliferation (mean: 209-fold), CFSE-labeling experiments demonstrated that the KLH-responsive population of postvaccination PBMCs represented 7-53% (mean: 23%). Combining CFSE-labeling with T-cell subset analysis confirmed the presence of CD4+ KLH-reactive T cells but also revealed a substantial population of CD8+ KLH-reactive T cells in one patient as well as minor populations of CD8+ KLH-reactive T cells in three other patients. Our data indicate that CFSE dye dilution analysis is a valuable tool for immune monitoring after dendritic cell vaccination.
Memory CD8 T cells mediate rapid and effective immune responses against previously encountered Ags. However, these cells display considerable phenotypic and functional heterogeneity. In an effort to identify parameters that correlate with immune protection, we compared cell surface markers, proliferation, and cytokine production of distinct virus- and tumor-specific human CD8 populations. Phenotypic analysis of epitope-specific CD8 T cells showed that Ag specificity is associated with distinct CCR7/CD45RA expression profiles, suggesting that Ag recognition drives the expression of these molecules on effector/memory T cells. Moreover, the majority of central memory T cells (CD45RAlowCCR7dull) secreting cytokines in response to an EBV epitope produces both IL-2 and IFN-gamma, whereas effector memory CD8 cells (CD45RAdullCCR7-) found in EBV, CMV, or Melan-A memory pools are mostly composed of cells secreting exclusively IFN-gamma. However, these various subsets, including Melan-A-specific effector memory cells differentiated in cancer patients, display similar Ag-driven proliferation in vitro. Our findings show for the first time that human epitope-specific CD8 memory pools differ in IL-2 production after antigenic stimulation, although they display similar intrinsic proliferation capacity. These results provide new insights in the characterization of human virus- and tumor-specific CD8 lymphocytes.
The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation.
Identification of reliable surrogate predictors for evaluation of cancer vaccine efficacy is a critical issue in immunotherapy. We analyzed quantitative and qualitative CD8+ T cell parameters in a large pool of BALB/c mice that were DNA-vaccinated against P1A self tumor-specific Ag. After immunization, mice were splenectomized and kept alive for a subsequent tumor challenge to correlate results of immune monitoring assays with tumor regression or progression in each individual animal, and to assess the prognostic value of the assays. The parameters tested were 1) percentage of in vivo vaccine-induced tumor-specific CD8+ T cells; 2) results of ELISPOT tests from fresh splenocytes; 3) percentage of tumor-specific CD8+ T cells in culture after in vitro restimulation; 4) in vitro increase of tumor-specific CD8+ T cell population expressed as fold of expansion; and 5) antitumor lytic activity of restimulated cultures. Except for the ELISPOT assay, each parameter tested was shown by univariate statistical analysis to correlate with tumor regression. However, multivariate analysis revealed that only in vitro percentage of Ag-specific CD8+ T cells was an independent prognostic factor that predicted tumor outcome. These findings should be considered in the design of new immune monitoring systems used in cancer immunotherapy studies.
Currently, there are numerous candidate HIV vaccines aimed at inducing T-cell mediated immune responses against HIV. To assess the immunogenicity of such vaccines, a reliable T cell assay must be utilized and typically one of the following assays is chosen for this purpose: bulk culture CTL, MHC I tetramer staining, IFN-gamma ELISPOT, or IFN-gamma intracellular cytokine staining. In this paper we report a comparison of the T cell responses detected by each assay in a large cohort of healthy normal volunteers vaccinated with adenovirus serotype 5 expressing HIV gag. Using stringently validated formats of each of these assays and pools of overlapping HIV gag peptides, we demonstrate that there is a high degree of correlation between all four of the common T cell assays, but inherent differences in the sensitivity of each assay to detect responders. In this study, the ELISPOT assay is shown to have the greatest sensitivity in detecting vaccine responses, while the ICS assay, although less sensitive, has the advantage of providing additional information on the phenotype of the responding cells.
Induction of peripheral tolerance in an antigen-specific manner is a critical goal of transplant biology. The specificity and avidity of multimerized peptide/major histocompatibilty complexes suggest their potential ability to modulate antigen-specific T-cell sensitization and effector functions.
A soluble divalent HLA-A2/IgG molecule (HLA-A2 dimer) was constructed and loaded with a self-protein origin peptide (Tyr(368-376)) to form a divalent Tyr/HLA-A2 molecule (Tyr/HLA-A2 dimer), which allowed for specific targeting to the epitope-specific cytotoxic T lymphocytes in bulk alloreactive T cells. Alloreactive T-cell response was induced by coculture of Tyr(368-376) -pulsed T2 cells (T2/Tyr) with peripheral blood lymphocytes of HLA-A2-negative (HLA-A2-ve) sample; five samples of HLA-A2-ve individuals were included in this study. After the coculture in the presence of Tyr/HLA-A2 dimer, the suppression of the dimer on alloresponse was characterized by analyzing allogeneic T-cell proliferation, specific cytolytic activity against the T2/Tyr, and specific Tyr/HLA-A2 tetramer staining.
The Tyr/HLA-A2 dimer suppresses alloreactive T-cell response by inhibiting its proliferation and cytotoxicity against specific target T2/Tyr in vitro, and it is interesting that the suppression is peptide-specific. The Tyr/HLA-A2 tetramer staining suggests the reduced function of CD8+ T cell is caused by inhibiting the generation of the epitope-specific alloreactive T cells by the Tyr/HLA-A2 dimer in three samples. Moreover, the existence of epitope-specific but function-negative T cells in the other two samples suggests that another mechanism might exist that is involved in silencing alloreactive responses by the dimer.
Peptide-loaded dimers offer a novel approach to induce peptide-specific immunosuppression and may be useful in promoting graft survival.
Veterinary species offer unique opportunities for the study of immune responses during natural host/pathogen interactions. Experimental studies can be used to characterize the response to infection, vaccination, and influence of vaccination on the response to infection. The intent of this review is to demonstrate the use of cell tracking dyes to monitor and characterize in vitro proliferative responses by mononuclear cell subsets from veterinary species as a correlate to the in vivo response. Selected examples are provided to illustrate the usefulness of this approach to characterize various tissue dendritic cell populations, CD8 alpha alpha(+) T cells, gammadelta T cells, and CD172a(+) cells. Comparative approaches provide unique and comprehensive insights into mononuclear cell biology that may be applicable to similarly described cell populations in humans.
The advent of contemporary digital instrumentation has enhanced both the potential and the complexity of flow cytometric experiments, allowing for the detailed dissection of immune cell subsets and their functions. The use of cell tracking labels such as PKH26 and CFSE has been important in observing such cellular functions, but their visible emission characteristics have limited the design of such analyses. As the demand for multiparametric flow cytometry intensifies, it will become increasingly important to utilize a broader range of cell tracking reagents to optimize the measurement of fluorescence signals and to provide flexibility in the use of commercially available fluorochrome - antibody combinations. We report on the evaluation of three lipophilic membrane dyes, CellVue Lavender, CellVue Plum and CellVue NIR780; with fluorescence emissions in the violet, far-red and near infrared wavelength regions, respectively. These reagents are similar to established tracking dyes such as PKH26 and CFSE in terms of staining procedure, membrane stability, optimal concentration, and lack of effect on cellular proliferation. The CellVue dyes however, exhibit different spectral characteristics than existing tracking compounds, and capitalize upon the increased number of lasers incorporated into commercially available instrumentation; thus permitting measurement of labeled populations in underexploited regions of the spectrum.
A better understanding of immune effector and regulatory pathways has led to innovative, and complex, immunotherapy strategies. CD8(+) cytolytic T lymphocytes (CTL) provide one common pathway of tumor cell destruction. The peripheral blood CTL compartment typically comprises a minority of anti-tumor CD8(+) lymphocytes and the determination of their number during clinical trials is the focus of various laboratory methods. We have monitored tumor specific CD8(+) as well as CD4(+) lymphocyte precursor frequencies in the peripheral blood using a Dye Dilution Proliferation Assay (DDPA). We summarize our experience applying DDPA in a multi-parameter, antigen-specific assay, detailing some of its complexities and advantages. We provide examples of our clinical trial results showing tumor-specific CD8(+) and CD4(+) precursor frequency (PF) data in patients being treated on novel immunotherapy trials.
Flow cytometric analyses of immune cell proliferation, differentiation, and function are limited by the number of different fluorochromes that can be resolved simultaneously. Additional colors to expand functional analytic capability will facilitate higher dimensional analyses of heterogeneous cell populations by basic and clinical scientists. Our aim in these studies was to evaluate CellVue Claret, a fluorescent, far-red emitting, membrane intercalating dye (excitation maximum: 655 nm, emission maximum 677 nm), as an alternative and/or complementary probe to PKH26 and CFSE(1) for polychromatic studies of immune cell proliferation and function. Using a BD FACSCalibur and human peripheral blood mononuclear cells (PBMCs) from 8 different donors (2 donors studied twice), we compared CellVue Claret with the two most commonly used visible-emitting proliferation dyes, PKH26 and CFSE, in terms of: (1) compatibility with 7-Amino-actinomycin D (7-AAD) as a viability marker; (2) effect of dye labeling on lymphocyte viability; and (3) the proliferative response of CD3+ T lymphocytes from 0-96 hours as assessed by dilution of each of the 3 cell tracking dyes in cultures stimulated with anti-CD3 plus IL-2. Post-labeling recoveries and viabilities were similar for all 3 dyes, with modestly higher initial staining intensities and coefficients of variation for CellVue Claret than for CFSE or PKH26. Lymphocyte viabilities in stimulated or unstimulated cultures were also unaffected by choice of dye. Proliferative responses of viable CD3+ lymphocytes were comparable for all three dyes, whether results were reported as Proliferative Fraction (percent of cells that had divided one or more times) or as Precursor Frequency (percent of parent population that had gone on to proliferate in response to anti-CD3 plus IL-2). In summary, T cell proliferation analysis using CellVue Claret gives results equivalent to those obtained with PKH26 or CFSE, expanding the choice of proliferation dyes suitable for use in high dimensional polychromatic studies on flow cytometers with far red (633 nm-658 nm) excitation capabilities.
Cell-tracking dyes stain cells with bright fluorescence which is partitioned between daughter cells after each cell division, so that the daughter cells have closely half the intensity of the parent cells from which they were derived. Therefore, the intensity of a cell, relative to its intensity at the time of staining, provides information about how many divisions have occurred. Knowing the number of division cycles that have occurred, one can calculate the number of cells in the original population (before culture) that were going to go on to proliferate. In this way, cell-tracking dyes provide a flow cytometric method for determining the proportion of cells in a population that will go on to proliferate in response to stimulation. This dye-dilution methodology can, therefore, detect proliferation of rare antigen-specific cells that increase in number during division and, importantly, can be used to back-calculate the precursor frequency of these rare cells. Simultaneously, the phenotype of these cells can be determined, as well as their ability to synthesize cytokines, and to express or not relevant antigen-binding receptors and activation markers.
The articles in this thematic issue, entitled "Tracking Cell Proliferation and Function," illustrate some of the choices made by authors pushing the envelope for cell tracking applications in their areas of interest. Over the past decade there has been a proliferation in the range of commercially available probes for these studies, the capabilities of the instrumentation used to detect them, and in the biological systems being studied. This introductory to the thematic issue presents the advantages and limitations of the more commonly used probes such as CFSE and PKH26, as well as emerging probes that expand the range of fluorescence available, including quantum dots and the new CellVue dyes. Appropriate method and instrument setup controls and possible data analysis strategies are discussed with the goal of urging experienced investigators to include all critical information and controls when publishing their data and of aiding researchers new to cell tracking to make informed decisions on which cell tracking reagent(s) are best suited for their particular application. All cell tracking assays have the common goal of determining the fate of a particular cell population within a heterogeneous environment, whether in vivo or in vitro. Some of the common themes among the contributions found in this issue include how various probes are used to track (i) cell proliferation, (ii) regulatory and effector immune cell function and (iii) membrane transfer and antigen presentation. Although these represent only a small fraction of the large and growing list of applications for cell tracking, clearly illustrate the growing trend toward the use of multiple tracking reagents and multiple detection modalities to address complex biological questions.
Naive T lymphocytes travel to T-cell areas of secondary lymphoid organs in search of antigen presented by dendritic cells. Once activated, they proliferate vigorously, generating effector cells that can migrate to B-cell areas or to inflamed tissues. A fraction of primed T lymphocytes persists as circulating memory cells that can confer protection and give, upon secondary challenge, a qualitatively different and quantitatively enhanced response. The nature of the cells that mediate the different facets of immunological memory remains unresolved. Here we show that expression of CCR7, a chemokine receptor that controls homing to secondary lymphoid organs, divides human memory T cells into two functionally distinct subsets. CCR7- memory cells express receptors for migration to inflamed tissues and display immediate effector function. In contrast, CCR7+ memory cells express lymph-node homing receptors and lack immediate effector function, but efficiently stimulate dendritic cells and differentiate into CCR7- effector cells upon secondary stimulation. The CCR7+ and CCR7- T cells, which we have named central memory (TCM) and effector memory (TEM), differentiate in a step-wise fashion from naive T cells, persist for years after immunization and allow a division of labour in the memory response.
We have developed a technology for analysis and sorting of live cells according to secreted molecules. An artificial affinity matrix, specific for the secreted product of interest, is created on the cell surface, and the cells are allowed to secrete for a defined time period. The secreted molecules bind to the affinity matrix on the secreting cell and are subsequently labeled with specific fluorescent or magnetic staining reagents for cytometric analysis and cell sorting. Crossfeeding of the secreted products to other cells is prevented by decreasing the permeability of the incubation medium. This approach will have a wide range of applications in biotechnology and biomedical research. Here, we describe analysis and sorting of hybridoma cells, according to secreted antibodies, and of activated T lymphocytes, according to secreted cytokines.
Identification and characterization of antigen-specific T lymphocytes during the course of an immune response is tedious and
indirect. To address this problem, the peptide-major histocompatability complex (MHC) ligand for a given population of T cells
was multimerized to make soluble peptide-MHC tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with
two different human immunodeficiency virus (HIV)-derived peptides or with a peptide derived from influenza A matrix protein
bound to peptide-specific cytotoxic T cells in vitro and to T cells from the blood of HIV-infected individuals. In general,
tetramer binding correlated well with cytotoxicity assays. This approach should be useful in the analysis of T cells specific
for infectious agents, tumors, and autoantigens.
A detailed understanding of the effects of costimulatory signals on primary T cell expansion has been limited by experimental approaches that measure the bulk response of a cell population, without distinguishing responses of individual cells. Here, we have labeled live T cells in vitro with a stable, fluorescent dye that segregates equally between daughter cells upon cell division, allowing the proliferative history of any T cell present or generated during a response to be monitored over time. This system permits simultaneous evaluation of T cell surface markers, allowing concomitant assessment of cellular activation and quantitative determination of T cell receptor (TCR) occupancy on individual cells. Through this approach, we find that TCR engagement primarily regulates the frequency of T cells that enter the proliferative pool, but has relatively little effect on the number of times these cells will ultimately divide. In contrast, CD28-costimulation regulates both the frequency of responding cells (particularly at sub-maximal levels of TCR engagement), and more prominently, the number of mitotic events that responding cells undergo. When CD28-stimulation is blocked, provision of IL-2 restores the frequency of responding cells and the normal pattern of mitotic progression, indicating that the other CD28-induced genes are not required for this effect. An unexpected finding was that even at maximal levels of TCR engagement and CD28-mediated costimulation, only 50-60% of the original T cells in culture can be induced to divide. The nondividing cells are heterogeneous for naive versus memory markers, suggesting a more complex relationship between expression of memory markers and the ability to be recruited into the dividing pool. From these studies, we conclude that a stringent checkpoint regulates the participation of activated T cells in clonal expansion, with TCR and CD28 signals having both overlapping and differential effects on the induction and maintenance of T cell responses.
Viral infections induce extensive T cell proliferation in vivo, but the specificity of the majority of the responding T cells has not been defined. To address this issue we used tetramers of MHC class I molecules containing viral peptides to directly visualize antigen-specific CD8 T cells during acute LCMV infection of mice. Based on tetramer binding and two sensitive assays measuring interferon-gamma production at the single-cell level, we found that 50%-70% of the activated CD8 T cells were LCMV specific [2 x 10(7) virus-specific cells/spleen]. Following viral clearance, antigen-specific CD8 T cell numbers dropped to 10(6) per spleen and were maintained at this level for the life of the mouse. Upon rechallenge with LCMV, there was rapid expansion of memory T cells, but after infection with the heterologous vaccinia virus there was no detectable change in the numbers of LCMV-specific memory CTL. Therefore, much of the CD8 T cell expansion seen during viral infection represents antigen-specific cells and warrants a revision of our current thinking on the size of the antiviral response.
Naive T lymphocytes have the potential to differentiate and produce a range of cytokines crucial for appropriate immune responses. How T lymphocytes vary their cytokine output during differentiation is unknown, although they are clearly influenced by the cytokines already present in the environment. Here we show that the number of divisions taken by the cells after activation is a critical element in T cell differentiation. Our experiments used the dye 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester to track cells in different divisions after activation by anti-CD3 in the presence of the differentiating cytokine interleukin (IL)-4. The patterns of acquisition or loss of secretion of IL-2, IL-3, IL-4, IL-5, IL-10, and interferon gamma all varied markedly with division number. These relationships were consistent regardless of the time-dependent variation in distribution of T cells among divisions. Thus, the observed combination of complex asynchronous T cell growth, overlaying a fixed probability of acquisition or loss of a cytokine at each division can explain why T cell differentiation displays the contradictory features of being both highly stochastic and highly controlled. Furthermore, these data reveal that T cells share a common regulatory strategy with B cells, whereby changes in the class of immune response are linked to the process of clonal expansion.
Comparative study of alloimmune responses against major and minor histocompatibility Ags has been limited by the lack of suitable assays. Here, we use a bioassay that permits tracking of alloreactive CD4+ T cell populations as they proliferate in response to major or minor histocompatibility Ags in vivo. Division of alloreactive CD4+ T cells proceeded more rapidly in response to major histocompatibility Ags than minor Ags, although CD4+ T cells alloreactive to minor Ags had a similar capacity to divide successively up to eight times after stimulation. Allorecognition of minor histocompatibility Ags was highly dependent on CD28 costimulation, with the frequency of CD4+ T cells proliferating in response to minor Ags in the absence of CD28 costimulation reduced up to 20-fold. These findings highlight differences in signaling processes that lead to allorecognition of major and minor histocompatibility Ags and have implications on the design of interventions aimed at abrogating these responses.
The adoptive transfer of TCR-transgenic T cells into syngeneic recipients allows characterization of individual T cells during in vivo immune responses. However, the proliferative behavior of individual T cells and its relationship to effector and memory function has been difficult to define. Here, we used a fluorescent dye to dissect and quantify T cell proliferative dynamics in vivo. We find that the average Ag-specific CD4+ T cell that undergoes division in vivo generates >20 daughter cells. TCR and CD28 signals cooperatively determine the degree of primary clonal expansion by increasing both the proportion of Ag-specific T cells that divide and the number of rounds of division the responding T cells undergo. Nonetheless, despite optimal signaling, up to one-third of Ag-specific cells fail to divide even though they show phenotypic evidence of Ag encounter. Surprisingly, however, transgenic T cells maturing on a RAG-2-/- background exhibit a responder frequency of 95-98% in vivo, suggesting that maximal proliferative potential requires either a naive phenotype or allelic exclusion at the TCRalpha locus. Finally, studies reveal division cycle-dependent expression of markers of T cell differentiation, such as CD44, CD45RB, and CD62L, and show also that expression of the cytokines IFN-gamma and IL-2 depends primarily on cell division rather than on receipt of costimulatory signals. These results provide a quantitative assessment of T cell proliferation in vivo and define the relationship between cell division and other parameters of the immune response including cytokine production, the availability of costimulation, and the capacity for memory.
We identified circulating CD8+ T-cell populations specific for the tumor-associated antigens (TAAs) MART-1 (27-35) or tyrosinase (368-376) in six of eleven patients with metastatic melanoma using peptide/HLA-A*0201 tetramers. These TAA-specific populations were of two phenotypically distinct types: one, typical for memory/effector T cells; the other, a previously undescribed phenotype expressing both naive and effector cell markers. This latter type represented more than 2% of the total CD8+ T cells in one patient, permitting detailed phenotypic and functional analysis. Although these cells have many of the hallmarks of effector T cells, they were functionally unresponsive, unable to directly lyse melanoma target cells or produce cytokines in response to mitogens. In contrast, CD8+ T cells from the same patient were able to lyse EBV-pulsed target cells and showed robust allogeneic responses. Thus, the clonally expanded TAA-specific population seems to have been selectively rendered anergic in vivo. Peptide stimulation of the TAA-specific T-cell populations in other patients failed to induce substantial upregulation of CD69 expression, indicating that these cells may also have functional defects, leading to blunted activation responses. These data demonstrate that systemic TAA-specific T-cell responses can develop de novo in cancer patients, but that antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth.
We studied the influence of memory T cell properties on the efficiency of secondary immune responses by comparing the in vivo immune response of the same numbers of T cell receptor (TCR) transgenic (Tg) naïve and memory T cells. Compared to naïve Tg cells, memory cells divided after a shorter lag time; had an increased division rate; a lower loss rate; and showed more rapid and efficient differentiation to effector functions. We found that initial naïve T cell priming resulted in cells expressing mutually exclusive effector functions, whereas memory T cells were multifunctional after reactivation, with each individual cell expressing two to three different effector functions simultaneously. These special properties of memory T cells ensure the immediate control of reinfection.
sensitive or provide more information than previously used assays. Importantly, some of these new techniques allow direct ex vivo analysis of T cells without in vitro amplification, thus providing a more accurate picture of the in vivo immune re- sponse. In this review, we describe some of the most widely used techniques for immune monitoring of specific T-cell responses. These various assays can be schematically divided into func- tional assays, which measure the secretion of a particular cy- tokine (ELISPOT and intracellular cytokines); assays which assess the specificity of the T cells irrespective of their func- tionality and which are based on structural features of the TCR (tetramers and immunoscope); and assays aimed at detecting T-cell precursors by amplifying cells that proliferate in re- sponse to antigenic stimulation. The sensitivity and immuno- logical relevance of these various methods are discussed. Ma- jor findings and future applications in basic and clinical immunology are also presented.
The rules that govern memory T cell differentiation are not well understood. This study shows that after antigenic stimulation naïve CD8+ T cells become committed to dividing at least seven times and differentiating into effector and memory cells. Once the parental naïve CD8+ T cell had been activated, this developmental process could not be interrupted and the daughter cells continued to divide and differentiate in the absence of further antigenic stimulation. These data indicate that initial antigen encounter triggers an instructive developmental program that does not require further antigenic stimulation and does not cease until memory CD8+ T cell formation.
Activation of CD8(+) cytolytic T lymphocytes (CTLs) by antigen is triggered by the interaction of clonotypic alphabeta T cell receptors (TCRs) with antigenic peptides bound to MHC class I molecules (pMHC complexes). Fluorescent multimeric pMHC complexes have been shown to specifically stain antigen-specific CTLs by directly binding the TCR. In tumor-infiltrating lymphocytes from a melanoma patient we found a high frequency of tyrosinase(368-376) peptide-specific cells as detected by IFN-gamma ELISPOT, without detectable staining with the corresponding A2/peptide multimers. Surprisingly, these T cells were able to lyse tyrosinase(368-376) peptide-pulsed target cells as efficiently as other specific T cells that were stained by multimers. Analysis of the staining patterns under different conditions of incubation time and temperature revealed that these results were explained by major differences in TCR-multimeric ligand interaction kinetics among the clones. Whereas no direct quantitative correlation between antigenic peptide concentration required for CTL effector functions and equilibrium multimer binding was observed interclonally, the latter was profoundly affected by the kinetics of TCR-ligand interaction. More importantly, our data indicate that similar levels of T cell activation can be achieved by independent CD8(+) T cell clonotypes displaying different TCR/pMHC complex dissociation rates.
Memory T lymphocytes proliferate in vivo in the absence of antigen maintaining a pool of central memory T cells (T(CM)) and effector memory T cells (T(EM)) with distinct effector function and homing capacity. We compared human CD4(+) naive T, T(CM), and T(EM) cells for their capacity to proliferate in response to cytokines, that have been implicated in T cell homeostasis. Interleukin (IL)-7 and IL-15 expanded with very high efficiency T(EM), while T(CM) were less responsive and naive T cells failed to respond. Dendritic cells (DCs) and DC-derived cytokines allowed naive T cells to proliferate selectively in response to IL-4, and potently boosted the response of T(CM) to IL-7 and IL-15 by increasing the expression of the IL-2/IL-15Rbeta and the common gamma chain (gamma(c)). The extracellular signal regulated kinase and the p38 mitogen-activated protein (MAP) kinases were selectively required for TCR and cytokine-driven proliferation, respectively. Importantly, in cytokine-driven cultures, some of the proliferating T(CM) differentiated to T(EM)-like cells acquiring effector function and switching chemokine receptor expression from CCR7 to CCR5. The sustained antigen-independent generation of T(EM) from a pool of T(CM) cells provides a plausible mechanism for the maintenance of a polyclonal and functionally diverse repertoire of human CD4(+) memory T cells.
The mechanisms by which immunological memory is maintained after infection or vaccination are still a matter of debate. Long-term survival of memory T cells does not require major histocompatibility complex (MHC) contact. We show here that compared with memory CD4+ T cells that maintain contact with MHC class II, memory CD4+ T cells deprived of MHC class II contact show distinct functional defects upon antigen re-encounter. Thus, in contrast to their survival, maintenance of the typical quality of memory T cells crucially depends on MHC-derived signals.
Recent work shows that after stimulation with antigen, CD4+ and CD8+ T cells embark on a programme of proliferation that is closely linked with the acquisition of effector functions and leads ultimately to memory-cell formation. Here, we discuss the signals required for commitment to this programme of development and the factors that might influence its progression. Models of the pathways of effector and memory T-cell differentiation are discussed, and we highlight the implications of this new understanding for the optimization of vaccine strategies.
In contrast with the low frequency of most single epitope reactive T cells in the preimmune repertoire, up to 1 of 1,000 naive CD8(+) T cells from A2(+) individuals specifically bind fluorescent A2/peptide multimers incorporating the A27L analogue of the immunodominant 26-35 peptide from the melanocyte differentiation and melanoma associated antigen Melan-A. This represents the only naive antigen-specific T cell repertoire accessible to direct analysis in humans up to date. To get insight into the molecular basis for the selection and maintenance of such an abundant repertoire, we analyzed the functional diversity of T cells composing this repertoire ex vivo at the clonal level. Surprisingly, we found a significant proportion of multimer(+) clonotypes that failed to recognize both Melan-A analogue and parental peptides in a functional assay but efficiently recognized peptides from proteins of self- or pathogen origin selected for their potential functional cross-reactivity with Melan-A. Consistent with these data, multimers incorporating some of the most frequently recognized peptides specifically stained a proportion of naive CD8(+) T cells similar to that observed with Melan-A multimers. Altogether these results indicate that the high frequency of Melan-A multimer(+) T cells can be explained by the existence of largely cross-reactive subsets of naive CD8(+) T cells displaying multiple specificities.
It is unclear why immunological control of HIV replication is incomplete in most infected individuals. We examined here the CD8+ T cell response to HIV-infected CD4+ T cells in rare patients with immunological control of HIV. Although high frequencies of HIV-specific CD8+ T cells were present in nonprogressors and progressors, only those of nonprogressors maintained a high proliferative capacity. This proliferation was coupled to increases in perforin expression. These results indicated that nonprogressors were differentiated by increased proliferative capacity of HIV-specific CD8+ T cells linked to enhanced effector function. In addition, the relative absence of these functions in progressors may represent a mechanism by which HIV avoids immunological control.
Memory T lymphocytes proliferate in vivo in the absence of antigen maintaining a pool of central memory T cells (T CM) and effector memory T cells (T EM) with distinct effector function and homing capacity. We compared human CD4 naive T, T CM , and T EM cells for their capacity to proliferate in response to cytokines, that have been implicated in T cell homeostasis. Interleukin (IL)-7 and IL-15 expanded with very high efficiency T EM , while T CM were less responsive and naive T cells failed to respond. Dendritic cells (DCs) and DC-derived cytokines allowed naive T cells to proliferate selectively in response to IL-4, and potently boosted the response of T CM to IL-7 and IL-15 by increasing the expression of the IL-2/IL-15R and the common chain (c). The extracellular signal regulated kinase and the p38 mitogen-activated protein (MAP) kinases were selectively required for TCR and cytokine-driven proliferation, respectively. Importantly, in cytokine-driven cultures, some of the proliferating T CM differentiated to T EM-like cells acquiring effector function and switching chemokine receptor expression from CCR7 to CCR5. The sustained antigen-independent generation of T EM from a pool of T CM cells provides a plausible mechanism for the maintenance of a polyclonal and functionally diverse repertoire of human CD4 memory T cells.
Binding fluorescent or radioactive reporter molecules to the lipid bilayer of cell membranes allows cell growth and trafficking to be monitored in vivo.
A previously unreported property of human mononuclear phagocytes is the ability of these cells to spontaneously aggregate. Fresh mononuclear cells obtained after plateletpheresis were noted to spontaneously form large cellular aggregates. Dual parameter immunofluorescence analysis demonstrated that the aggregating cells were positive for the monocyte marker CD11 (complement receptor, type 3) but were negative for the lymphocyte marker CD3 (T3 antigen). In addition, less than 5% of the nonaggregating cells were CD11+, suggesting that almost all CD11+ cells aggregated. Cellular aggregates were independent of cell concentration and formed more efficiently at 4 degrees C than at either 22 or 37 degrees C. Based on these observations, a purification procedure utilizing Ficoll-Hypaque separation, spontaneous aggregation at 4 degrees C, and transient plastic adherence resulted in a sevenfold enrichment of the CD11+ peripheral blood monocytes. Purified monocytes were contaminated with less than 2% CD3 cells. The size, growth, and adherence characteristics as well as cytologic stains indicated that the monocytes were not significantly altered by the purification procedure. Thus, spontaneous aggregation is an efficient and convenient method for the isolation of large numbers of purified monocytes.
Techniques currently available for determining cell division are able to show one or, at best, a limited number of cell divisions. Other methods exist which can quantify overall division, but tell nothing about the division history of individual cells. Here we present a new technique in which an intracellular fluorescent label is divided equally between daughter cells upon cell division. The technique is applicable to in vitro cell division, as well as in vivo division of adoptively transferred cells, and can resolve multiple successive generations using flow cytometry. The label is fluorescein derived, allowing monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes to be used to immunophenotype the dividing cells.
During the last years it has become increasingly clear that production of most cytokines is not confined to one cell type. Thus, a method to detect cytokines at the single cell level would be a helpful tool to study the contribution of different cells to cytokine production in heterogeneous cell populations. Recently, Sander et al. (1991) demonstrated that it is possible to detect intracellular cytokines by fixation with paraformaldehyde, permeabilization with saponin and subsequent indirect immunofluorescent staining using fluorescence microscopy. Here, we describe a modified method to increase the specific intracellular staining which enables us to detect IFN-gamma, IL-2 and IL-4 producing cells by single laser flow cytometry. The carboxylic ionophore monensin was used to interrupt intracellular transport processes leading to an accumulation of the cytokine in the Golgi complex. This resulting increase of the signal/noise ratio permitted us to detect weakly fluorescent cells such as IL-4 producing cells. While IL-4 was detected in approximately 1-3% of peripheral mononuclear cells from healthy donors, up to 30% of the cells produced IFN-gamma and nearly 50% IL-2 after phorbol ester and ionomycin stimulation. Microscopic and flow cytometric analysis showed a highly significant correlation. Using three-color flow cytometry it was possible to measure intracellular cytokines and cell surface markers simultaneously. Subpopulations of human T cells (e.g., CD4+ CD45R0-) producing a restricted cytokine pattern could be identified by cell surface staining and were characterized by their cytokine production. Consequently, there was no further need for cell sorting to determine cytokine producing subsets in heterogeneous cell populations. We have tested human T cell clones for intracellular cytokine production and found a high concordance to ELISA analysis of the supernatants. We conclude that detection of intracellular cytokines by flow cytometry is a rapid, easy and semiquantitative assay which may be used to study individual cells in heterogeneous populations as well as to screen homogeneous cells for their cytokine pattern. This method is particularly relevant in view of the accumulating evidence of the functional role that subsets of (T) cells may play in various diseases depending on the pattern of cytokines they produce.
Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and lymphokine-activated killer cells has been demonstrated to mediate regression of tumors in murine models and in selected patients with advanced cancer. Improved methods for monitoring immune cell traffic, particularly to sites of tumor, are needed to elucidate mechanisms of antitumor activity and optimize treatment protocols. Traditional cell tracking methods such as fluorescent protein labeling and radiolabeling using 111In, 125I, or 51Cr are limited by isotope half-life, leakage or transfer of label from immune cells, and toxicity or altered cell function caused by the labeling process. Labeling with genetic markers allows long-term cell tracking but is laborious to perform and difficult to quantitate. We have used two recently described lipophilic cell tracking compounds (PKH26 and 125I-PKH95) which stably partition into lipid regions of the cell membrane to track immune cells in vivo. Concentrations of each tracking compound which had no adverse effects were determined for a variety of murine TIL and lymphokine-activated killer cell functions. Viability was unimpaired at labeling concentrations of up to 5 microM for PKH95 and 20 microM for PKH26. TIL proliferation was unaltered by labeling with up to 5 microM PKH95, 20 microM PKH26, or a combination of 15 microM PKH26 and 5 microM PKH95. In vivo cytotoxic effector function and in vivo therapeutic efficacy of lymphokine-activated killer cells and TIL were also unimpaired by labeling with 20 microM PKH26 or 1 microM 125I-PKH95. Subsequent studies in an adoptive transfer immunotherapy model used 125I-PKH95 to track the biodistribution of TIL in tumor and in non-tumor-bearing animals and PKH26 fluorescence to monitor microdistribution within tissues and distinguish TIL from host T-cells. The results suggest that differential accumulation, selective retention, or proliferation at the tumor site cannot account for the observed pattern of therapeutic efficacy. We hypothesize that a minimum number of TIL must reach the tumor site in order to achieve a demonstrable therapeutic effect.
We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8⁺ T cells recognizing peptide antigens presented by HLA-A2.1. CD8⁺ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 × CEM.T2 cells. Tumor necrosis factor α (TNF-α) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-α antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1.
We have used PKH26 dye, which is incorporated stably into the membrane of cells, to determine, using flow cytometry, lymphocyte proliferative responses to the antigen tetanus toxoid in fresh and cryopreserved samples. Measuring cell proliferation with this dye has advantages over either 3H-thymidine or Bromodeoxyuridine (BrdU). Whereas the existing methods measure proliferation at a single time point, PKH26 gives a cumulative measure of cell proliferation. As PKH26 is incorporated into the cell membrane, cells do not have to be permeabilised to allow dye incorporation into a cytoplasmic compartment. Most importantly, PKH26 can be used in combination with monoclonal antibodies to surface markers on mixed populations of cells, to determine the proliferation of individual subpopulations, without the need for prior cell fractionation. We also show that PKH26 can be used with similar efficacy in both fresh and cryopreserved samples. In addition since PKH26 is a cumulative measure of proliferative responses we were able to show that restimulation of the dividing population in vitro with fresh antigen presenting cells (APC) and antigen permits characterisation of a further proliferating cell population. The use of PKH26 dye in combination with cell phenotyping and measurement of cytokine production at the single cell level will prove a powerful tool for multiparameter analyses of cellular responses to antigen.
Helper T (Th) cell differentiation is highly regulated by cytokines but initiated by mitogens. By examining gene expression in discrete generations of dividing cells, we have delineated the relationship between proliferation and differentiation. Initial expression of IL-2 is cell cycle-independent, whereas effector cytokine expression is cell cycle-dependent. IFNgamma expression increases in frequency with successive cell cycles, while IL-4 expression requires three cell divisions. Cell cycle progression and cytokine signaling act in concert to relieve epigenetic repression and can be supplanted by agents that hyperacetylate histones and demethylate DNA. Terminally differentiated cells exhibit stable epigenetic modification and cell cycle-independent gene expression. These data reveal a novel mechanism governing Th cell fate that initially integrates proliferative and differentiative signals and subsequently maintains stability of the differentiated state.
It is commonly accepted that a high level of antigen specificity is a feature of T-cell activation. However, here, Don Mason argues that a comprehensive response to foreign antigens requires that T cells are widely crossreactive, such that one cell reacts productively with approximately 106 different MHC-associated minimal peptide epitopes.
Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.
The present study describes the optimization of an in vitro culture method for generating large amounts of dendritic cells (DC) in serum-free conditions from leukapheresis containing a mixed population of peripheral blood mononuclear cells (PBMC) which are cultured in the presence of GM-CSF and IL-13. Initial comparisons between the generation of DC from bulk and monocyte-enriched leukapheresis products showed that the presence of lymphocytes during the culture favors the differentiation of monocytes into DC. DC yields obtained from mixed mononuclear cell cultures were between 38 and 54% higher than yields obtained from monocyte-enriched cultures. Both types of cultures resulted in the generation of DC with an immature phenotype (CD83- and high phagocytic activity), which have been previously shown to be good stimulators for T cell responses. DC yields of bulk cultures in serum-free conditions were significantly higher than those obtained in the presence of 2% human serum. The cytokines of the supernatants of serum-free cultures comprised a significant content of pro-inflammatory cytokines such as IL-1, IL-12 and TNF-alpha. Maturation of DC generated by this method can be induced by treatment with double-stranded RNA, LPS or TNF-alpha, resulting in enhanced surface expression of CD80, CD86, CD40, CD83 and MHC molecules on the DC. The methodology described here offers the possibility for generating large amounts of clinical grade DC from bulk leukapheresis products, thus avoiding DC precursor purification steps, and thereby minimizing the risks of contamination. This culture process may be applied to cell-based therapeutic approaches for the treatment of cancer or chronic viral infections.
Since its introduction in 1994 (J. Immunol. Methods 171 (1994) 131), the flow cytometric analysis of lymphocyte proliferation by serial halving of the fluorescence intensity of the vital dye CFSE (carboxyfluorescein diacetate, succinimidyl ester or CFDA-SE) has become widely used in immunological laboratories around the world. This technique allows the visualisation of eight to 10 discrete cycles of cell division by flow cytometry, both in vitro and in vivo. Appropriately conjugated antibodies can be used to probe surface marker changes as cells divide, or changes in expression of internal molecules such as cytokines when appropriate fixation and permeabilisation protocols are used. An added advantage of the technique is the ability to recover viable cells which have undergone defined numbers of cell divisions by flow cytometric sorting, allowing functional studies to be performed. Other commonly used assays of cell proliferation give only limited information, as they usually measure division at a population level. The CFSE technique can be used to determine kinetics of immune responses, track proliferation in minor subsets of cells and follow the acquisition of differentiation markers or internal proteins linked to cell division.
The identification of distinct T helper lymphocyte subsets (Th1/2) with polarised cytokine production has opened up new fields in immunobiology. Of the several alternative methods of monitoring cytokine production, flow cytometric analysis of intracellular staining has distinct advantages and pitfalls. It allows high throughput of samples and multiparameter characterisation of cytokine production on a single cell basis without the need for prolonged in vitro culture and cloning. However, these methods may cause important changes in cell surface phenotype which can make interpretation difficult.
The immune response is initiated in organized lymphoid tissues where antigen-loaded dendritic cells (DCs) encounter antigen-specific
T cells. DCs function as packets of information that must be decoded by the T cell before an appropriate immune response can
be mounted. We discuss how the dynamics of DC–T cell encounter and the mechanism of T cell differentiation make the decoding
of this information stochastic rather than determinate. This results in the generation of both terminally differentiated effector
cells and intermediates that play distinctive roles in protection, immunoregulation, and immunological memory.
Understanding the lineage differentiation of memory T cells is a central question in immunology. We investigated this issue by analysing the expression of the chemokine receptor CCR7, which defines distinct subsets of naive and memory T lymphocytes with different homing and effector capacities and antiviral immune responses to HIV and cytomegalovirus. Ex vivo analysis of the expression of CD45RA and CCR7 antigens, together with in vitro analysis of the cell-division capacity of different memory CD8+ T-cell populations, identified four subsets of HIV- and CMV-specific CD8+ T lymphocytes, and indicated the following lineage differentiation pattern: CD45RA+ CCR7+ --> CD45RA- CCR7+ --> CD45RA- CCR7- --> CD45RA+ CCR7-. Here we demonstrate through analysis of cell division (predominantly restricted to the CCR7+ CD8+ T-cell subsets) that the differentiation of antigen-specific CD8+ T cells is a two-step process characterized initially by a phase of proliferation largely restricted to the CCR7+ CD8+ cell subsets, followed by a phase of functional maturation encompassing the CCR7- CD8+ cell subsets. The distribution of these populations in HIV- and CMV-specific CD8+ T cells showed that the HIV-specific cell pool was predominantly (70%) composed of pre-terminally differentiated CD45RA- CCR7- cells, whereas the CMV-specific cell pool consisted mainly (50%) of the terminally differentiated CD45RA+ CCR7- cells. These results demonstrate a skewed maturation of HIV-specific memory CD8+ T cells during HIV infection.
In defense of the host, the immune system must often raise an effective cytotoxic T lymphocyte (CTL) response from a small number of clonal precursors. The degree to which activation stimuli regulate the expansion and differentiation of naïve CTLs, however, remains unknown. Using an engineered antigen-presenting cell (APC) system that allows control over antigenic stimulation, we studied the signaling duration requirements for priming and clonal expansion of naïve CTLs. We found that naïve CTLs become committed after as little as 2 h of exposure to APCs and that their subsequent division and differentiation can occur without the need for further antigenic stimulation of the daughter cells, whether priming is in vitro or in vivo. These data show that after a brief interaction with stimulatory APCs, naïve CTLs initiate a program for their autonomous clonal expansion and development into functional effectors.
The development of soluble tetrameric MHC/peptide complexes has opened the possibility to directly identify and monitor antigen-specific CD8+ T cells in different clinical situations. This represents a technological breakthrough for the field of cell-mediated immunity. For example, the direct identification and enumeration of tumor-specific CD8+ T cells at the tumor site and in blood has recently provided compelling evidence that strong anti-tumoral responses naturally occur in some cancer patients. Moreover, the use of tetramers plays an essential role in the design of vaccination protocols aimed at inducing a strong and protective CD8+ T cell-mediated anti-tumoral response in cancer patients. The monitoring of antigen-specific T cell responses elicited by various peptide-based vaccines tested in phase I clinical trials clearly indicates that tumor-specific CD8+ T cells can be activated effectively at least in some cancer patients. Thus, multiparameter monitoring of antigen-specific T cell responses that combines ex vivo tetramer staining with various phenotyping and functional assays provides a novel approach to assess the functional potential of tumor-specific T lymphocytes and may also facilitate the optimization of vaccination protocols.
A major challenge in Phase I clinical trials of cancer vaccines is how to identify the optimal regimen and/or dosing for Phase II and III trials. Immunologic responses to tumor-associated antigens (TAA) could potentially serve as surrogate markers. Assays which detect cytokine production and/or secretion by antigen (Ag)-reactive T cells are increasingly being used to monitor such responses in clinical trials. A potential problem of these assays is high non-specific background and/or inability to easily identify the T-cell phenotype. We designed an assay, termed cytokine-specific affinity matrix (Multi-CSAM) assay,(1) which can determine the frequencies of phenotypically identifiable antigen (Ag)-reactive T cells. The assay relies on the tightly regulated production and secretion of cytokine by Ag-activated cells, capture of secreted cytokine by an artificial cytokine-specific cell-surface affinity matrix and flow-cytometric detection of bound cytokine with a fluorochrome-labeled anti-cytokine mAb. In an attempt to minimize non-specific background, the assay excludes non-viable cells by using the cell-impermeant DNA-binding dye TO-PRO-3, and selects activated cells on the basis of CD69 expression. Furthermore, the phenotype of the Ag-reactive T cells is identified using the CD4 and CD8 T-cell markers. Thus, using an appropriately modified two-laser benchtop instrument, a single sample can be evaluated by five-color multiparametric flow-cytometry that employs a combination of appropriately labeled mAbs. The five colors selected are anti-IFNgamma-PE, the dye TO-PRO-3, anti-CD69-PerCP, anti-CD4-FITC and anti-CD8-PE/Texas Red. Although our ultimate goal is to identify T cells reactive to TAA, as a first step this report demonstrates that Multi-CSAM can be used to detect the reactivity to TT and Candida.
Identification of MHC-restricted antigens and progress in the induction and control of adaptive cytotoxic immune responses have led to renewed interest in immunotherapy as a treatment for severe pathologies such as cancer and autoimmune diseases. Reliable procedures for detecting and monitoring T cell responses induced by the treatment throughout a clinical trial are needed in order to design rational protocols with increased efficiency. We have attempted to develop such a procedure by combining T cell sorting using HLA-peptide complexes multimerized on magnetic beads together with the quantitative Immunoscope approach. Once a recruited patient has been typed for HLA and target antigens, relevant HLA--peptide multimers can be selected and used for sorting specific peripheral T cells prior to any treatment and at the peak of the expected response to treatment. Clonotypic primers specific for the TCR rearrangements of the specific T cell clones can then be designed and used for measuring the frequency of their TCR transcripts by quantitative PCR on blood samples or T cell subsets throughout the trial. In reconstruction experiments as well as in samples from one rheumatoid arthritis patient, we were readily able to detect and follow several T cell clones with a frequency as low as 10(-5) among CD8+ T cells. The main advantages of this procedure over other currently available assays are that it does not require any assumptions on the functional status of the specific T cells and it permits the monitoring of individual T cell clones whose phenotypic shift can thus be evaluated.
Immunologic memory results from a carefully coordinated interplay between cells of the immune system. In this review, we explore various aspects of the nature, generation, and maintenance of T lymphocyte-mediated immunologic memory. In light of the demonstrated heterogeneity of the memory T-cell pool, we hypothesize that subsets of memory T cells instructed to mature to distinct differentiation stages may differ, not only in functional and homing properties, but also in the conditions they require for survival, including antigen persistence and cytokine environment. Hence, according to this hypothesis, distinct memory T-cell subsets result from the nature and timing of the signals provided by the immune environment and occupy distinct niches. Intracellular and extracellular molecular mechanisms that underlie and modulate T-cell memory are discussed.
To understand the success or failure of immune responses against pathogens or tumours requires the direct measurement of specific lymphocytes. Recently, there has been an explosion of data in this field through the use of several new tools for measuring the number and function of T cells. This has allowed immunologists who study human disease and mouse models of infection and cancer to readily track specific T cells--in both time and space. Although there are common patterns, over time, each host-pathogen relationship seems to develop unique characteristics, as reflected in the quality of the T-cell response.
Peptide-bound histocompatibility leukocyte antigen (HLA) class I tetramers enable a precise identification of antigen-specific CD8+ T cells using flow cytometry. The combination of this technology with intracellular staining techniques opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). In this review, we hope to provide an overview of these possibilities, as well as making specific suggestions about the use of intracellular staining techniques in the study of antigen-specific T cells. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of our cellular immune system.
Memory T lymphocytes divide in vivo in the absence of antigen maintaining a pool of central memory (T(CM)) and effector memory cells (T(EM)) with distinct effector function and homing capacity. We compared human CD4+ naïve T, T(CM) and T(EM) cells for their capacity to proliferate in response to cytokines, which have been implicated in T cell homeostasis. Interleukin (IL)-7 and IL-15 expanded with very high efficiency T(EM), while T(CM) were less responsive and naïve T cells did not respond at all. Dendritic cell (DC)-derived cytokines allowed naïve T cells to respond selectively to IL-4 and potently boosted the response of T(CM) to IL-7 and IL-15 by increasing the expression of the IL-2/IL-15Rss and the common gamma chain (gamma(c)). The ERK and the p38 MAP kinases were selectively required for TCR and cytokine-driven proliferation, respectively. Importantly, in cytokine-driven cultures T(CM) proliferated and some of the proliferating cells acquired effector function and non-lymphoid tissue homing capacity. Ex vivo BrdU incorporation experiments showed that both T(CM) and T(EM) proliferated under steady state conditions in vivo. Altogether these results provide a plausible mechanism for the maintenance of a polyclonal and functionally diverse repertoire of human CD4+ memory T cells and for a sustained antigen-independent generation of T(EM) from a pool of T(CM) cells.
Department of Physiology
- Corresponding
* Corresponding author. Department of Physiology, Dartmouth Medical School, Lebanon, NH 03756, USA. Tel.: +1-603-650-7661; fax: +1-603-650-6130.
Determination of lymphocyte division by flow cytometry
- Lyons
Following the fate of individual T cells throughout activation and clonal expansion. Signals from T cell receptor and CD28 differentially regulate the induction and duration of a proliferative response
- Wells
Effector and memory T-cell differentiation: implications for vaccine development
- Kaech
Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients
- Lee